The present invention relates generally to a cassette reader and, more specifically, to a colorimeter for detecting the status of stained and/or unstained samples, to quantify changes in sperm cell color density and motility.
Colorimeters are generally used to characterize the color of an object by providing diffuse illumination which is reflected or transmitted by the object and is transmitted optically to a dispersing element. The dispersing element disperses the coherent light spectrally. A normal colorimetry is effected by using a spectrophotometer. However, the spectrophotometer is a large and expensive apparatus, which can not be readily used in general and does not facilitate use outside a laboratory environment for determining the status of stained samples such as biological specimens.
Another problem associated with calorimeters is that as the illuminating lamps used in such colorimeters (for example the halogen lamps) age, the color and intensity of light produced by the lamp can change. This in turn, causes the color signature of a sensed object to vary with the age of the lamp. A possible solution to the problem of lamp aging is frequent recalibration of the colorimeter. However, this is only feasible in the laboratory environment and frequent recalibration increases the cost of assaying samples.
Bolan et al., U.S. Pat. No. 4,952,817, issued Aug. 28, 1990, describes a self-starting test station controlled by a microprocessor and includes a slot guiding a strip carrying a sample. The essential feature of the Bolan patent is that it is always xe2x80x9creadyxe2x80x9d, and turns on only when it senses a test sample inserted. It is battery powered, with a battery powered computer. The power is normally off, there is no power to the microprocessor and negligible power elsewhere. Bolan""s invention is directed at blood sugar monitoring for diabetics. A separate calibration stick measures the blood sugar content. The disadvantage of this system is that by having a separate calibration stick, the analysis of each sample is open to error. The present invention increases the accuracy by providing the calibration on the cassette so that each measurement is made with a built-in scale on each cassette.
Stark, E. W., U.S. Pat. No. 5,433,197, issued Jul. 18, 1995 describes an apparatus to measure glucose in the eye. This depends on transmission of light through the eye. The disadvantage is that it requires use of a diffraction grating in either the out-put light or the light analyzing sections to give narrow light beams. It uses infra-red illumination exclusively and obtains an absorption spectrum. Therefore, the unit cannot measure the reflected intensity of a cassette. The present invention uses only visible illumination and therefore integrates the absorption without requiring spectral measurement.
Heretofore, the analysis of biological, industrial and medical specimens has been accomplished by determining the concentration of a dye or dyed cells in a specimen by conventional colorimetry. This method of color differentiation suffers from several drawbacks. The process is slow and cumbersome, requires costly equipment which needs frequent calibration in a laboratory environment and is unsuitable for use in other environments such as a doctor""s office or even an industrial environment.
By way of example, over 11 million semen analyses are performed annually worldwide, many of them in clinical practices that can not afford fully automated semen analysis systems or colorimeters. The most important features of the semen analysis systems are determination of sperm concentration, motility, viability, white cell count, morphology and acrosome reactions. A semen analysis may be performed for a variety of reasons, including not only suspected infertility, but quality controls for sperm banks, male contraceptive research and post vasectomy checks for sperm counts. In the U.S., 500,000 vasectomies are performed per year, and in China and India, the total exceeds 3.5 million annually (Marquette, C. M. et al., 1995, Am. J. Public Health 85: 644-649). Two to three follow-up tests are recommended in the weeks after surgery to ensure that no live sperm are left in the system.
At present, semen is examined either visually by trained observers or with an automated semen analysis system. The World Health Organization guidelines for semen analysis require numerical information on semen criteria such as sperm concentration and counts, motility, viability, morphology and presence of leukocytes in order to determine the course of treatment for infertility. In some countries, for example, Germany, the cost of yes/no tests is not reimbursable. Only cost of tests producing results having a numerical read-out is reimbursed. In consequence, sperm cells are counted in a hemacytometer, viability determined by further staining and counting on a slide and morphology determined by trained technicians. It is therefore desirable to have an inexpensive simple method to cut costs while producing numerical measurements of semen criteria. There is a need for an inexpensive, accurate method for determining key elements, including quantifying sperm counts, in male fertility assessments.
In addition, there is a need to more inexpensively measure sperm motility in male fertility assessments. Previous attempts to use optical scattering and laser Doppler shift to measure sperm motion and concentration have met with limited success (Brotherton J., 1988, Andrologia 20:33-43; Schirren, S. et al., 1988, Andrologia 20:44-47). These approaches used high concentrations of sperm in relatively deep chambers and relied on statistical properties of large numbers of sperm. Woolhouse, J. K. et al., U.S. Pat. No. 4,601,578, issued Jul. 22, 1986, (abandoned) discovered that laser light scattered from a semen sample contains information indicative of the motility of the sperm contained in the sample, not by virtue of the moving sperm causing Doppler Shifts in the light frequency but by virtue of the flat sperm heads simulating mirrors which rotate at a rate proportional to sperm beat frequency. The laser light scattered from a sperm sample was subjected to Fourier analysis in an attempt to derive both the motile and static sperm concentrations. However, in order to evaluate the signal spectrum the output signal had to be split into two frequency bands and integrated the signals with respect to time to provide a reasonable measure of the amplitude. Moreover, such an integration technique requires an accurately determined average or DC signal level which posed a significant problem requiring use of a high pass master filter. Rigler, R. U.S. Pat. No. 5,116,125, issued May 26, 1992, described a method to measure laser scattering from a sample in a cylindrical capillary tube. The disadvantage of this method is that the method requires equipment which is very expensive, non-portable and provides more information than is needed for routine work associated with male fertility assessments.
The present assays are based on staining sperm cell nuclei, acrosomes and leukocyte cytoplasm in order to detect only the stained cells rather than both cells and detritus. A new method of determining cell motility is also developed using a focused laser beam which measures swimming sperms by laser light scattering and provides a ratio of motile to total cell density of the specimen. Trends in medical cost containment are driving the need for low cost rapid diagnostics simple enough to be used in the general practitioner""s office. Accordingly, it is desired to provide a calorimeter for determining sample characteristics that is inexpensive and suitable for automatic quantification of semen (or other tested sample) properties.
In accordance with the instant invention, a cassette reader or male assessment of reproductive quality (MARQ) reader measures the intensity of a light beam reflected off a stained test specimen. The calorimeter includes a housing. Two or more high power light emitting diodes are disposed within the housing for shining light along a light path to the sample. A detector detects light reflected from the sample. A microprocessor receives the reflected light and determines the characteristic of the specimen based thereon, and reports the result(s) on a display.
The housing is provided with a cover having a guiding slot and a plurality of position detectors which detect the position of a sample holder member when the sampler holder is placed within the guiding slot. A mirror assembly for maximizing light transmitted to the sample is disposed between the light source and samples.
Accordingly, it is an object of the invention to provide a cassette reader which is simple to operate and which better maintains its accuracy over time.
Another object of the invention is to provide a cassette reader adapted to assay the male assessment of reproduction quality (MARQ) in testing male fertility.
Another object of the invention is to provide a cassette reader which selectively provides a plurality of position detectors which detects the position of a sample holder member within the card reader.
Another object of the invention is to provide a cassette reader having high power light emitting diodes in the color regions appropriate for measurement of the visual stain on the test specimen.
Another object of the invention is to provide a cassette reader having high power light emitting diodes in the color regions appropriate for measurement of the unstained test specimen.
Another object of the invention is to provide a cassette reader which permits the illuminating light to penetrate on to the sample while directing the scattered light on to its focal point at the detector.
Still another object of the invention is to provide a cassette reader having a lens which further maximizes the surface reflection of the incident illuminating beam on to the detector.
Another object of the invention is to provide a cassette reader which permits accurate determination of motile cells in a sperm specimen.
Still another object of the invention is to provide a cassette which is arranged to hold a small microscope slide of fixed chamber depth.
Another object of the invention is to provide a cassette reader having laser illumination which is focused by a condensing lens on to a sample chamber.
Another object of the invention is to provide a cassette reader having a sensing means including an apertured-mirror optical system to determine light scattering by a motile sperm.
Still other objects and advantages of the invention will in part be obvious and will in part be apparent from the specifications and drawings.
The invention accordingly comprises the features of construction, combination of elements, and arrangement of parts which will be exemplified in the constructions hereinafter set forth, and the scope of the invention will be indicated in the claims.